The human CKs include four proteins encoded by different genes. Two proteins that are derived from the cytoplasm, and are called ‘muscle type (M type)’ and ‘brain type (B type)’ depending on their localization, and others that are derived from the mitochondria. CK isozymes derived from the cytoplasm are dimers consisting of M type and/or B type, and are classified as CK-MM, CK-MB, and CK-BB. Mitochondria CKs (mCKs) are octamers, and two kinds exist very stably, but they dissociates into dimers within a few minutes in the presence of a transient state-analogue complex consisting of creatine, Mg, ADP and nitrate. It is known that they gradually dissociate in the blood (Karin Fritz-Wolf et al., Nature, 361, 341–345 (1996)).
These isozymes migrate in the electrophoresis from the cathode side in the following order: mCK (octamer)>mCK (dimer)=CK-MM>CK-MB>CK-BB. The mCK (dimer) has a mobility equal to that of CK-MM, so that the former can be mis-identified as ‘CK-MM’ in the preserved blood. The macro CK bound to immunoglobulin also exits although it is not an isozyme. These can be identified from a zymogram by the mobility, the immunity counter current method, and so on.
The assay of CK and CK isozymes is widely used in the clinical test. CK-MB is important as a marker for the cardiac infarct among them. CK-MB is assayed by the EIA method, the immunity inhibitation, the electrophoresis method, and so on. Although the EIA method permits specifically assaying only CK-MB, it requires a specially designed instrument, and has a problem in the promptness. The electrophoresis method requires a complex manipulation and skill, and a densitometer is necessary to obtain a ratio of CK-MB, i.e., the method also has a problem in the promptness. Although the immunity inhibitation permits a rapid and simple assay when an automated analyzer is used, it is a fault that it lacks the specificity.
Under the present situation, however, it is required to diagnose the cardiac infarct at an early stage. Therefore, the immunity inhibitation that permits the rapid and simple assay is widely used. In this method, the enzyme activity of CK-M subunit is inactivated using a specific antibody against the CK-M subunit (‘anti-CK-M antibody’), and remaining CK-B subunit activity is assayed. By this method, CK-BB and mCK (dimer+octamer) as well as CK-MB are also assayed. Among these, little CK-BB is present in the blood, i,e,, negligible. Although diseases caused by the deviation of CK-BB include the brain contusion and the multiple organ failure, these cases are rare. Even in the serum of a healthy person, mCK is contained in an active amount nearly equal to CK-MB (Yoko Toyoda et al., Seibutsu Butsurikagaku, 41, 244 (1997); Tadashi Hoshino et al., Seibutsu Butsurikagaku, 42 (supplement) 2, 21 (1998)). In addition, in the cell necrosis such as liver disease and the malignant tumor, mCK is deviated, and the judgment of the result is disturbed. Recently, it was reported that mCK is deviated by the enteritis by rotavirus and the asphyxia neonatorum (Tadashi Hoshino et al, Rinsho Byori 46, Meeting Issue, 57 (1998); Fusae Kanemetsu et al, Rinsho Byori 46, Meeting Issue, 58 (1998). It was also reported that the presence of mCK affects CK-MB measurements by the enzyme inhibition method (Abstracts for 22nd Meeting of The Chiba Pref. Society for Clinical and Hygienic Tests 6—7 (February 2001). Then, a method was reported in which an antibody specific to mCK activity(anti-mCK antibody) was prepared and anti-mCK antibody together with anti-CK-M antibody was made to act to CK isozymes to inhibit CK-M subunit activity and mCK activity for more accurate assay of CK-MB (JP P2002-270A).
The mCK includes ubiquitous mCK (umCK) and sarcomeric mCK (smCK) isoforms. It was reported from the genetic analysis of mCK that amino acid sequences of human mCK, CK-M and CK-B are homologous one another at 62–66% and that amino acid sequences of umCK and smCK are homologous each other at 80% (J. Biol. Chem. 265: 6921–6927 (1998)). Isolating umCK and smCK and examining the properties of them revealed that umCK and smCK form dimer and octamer, respectively, and have slightly different pI values and that the octamer gave antigenicity at a similar level and was discriminated from the dimer (Fusae Kanemitsu et al., Rinsho Byori 47, Meeting Issue, 306 (1999)).
The relation between the deviation of mCK and diseases has been frequently reported very often as described above. The mCK is generally assayed by the agarose electrophoresis, and umCK and smCK behave similarly, so that both have been reported without distinguishing each other. Although an antibody having an anti-smCK activity was reported (JP P2002-270A), no report was published concerning anti-umCK antibody, and it was difficult to easily distinguish and analyze both.